Apoptotic protease-activating factor 1 (Apaf-1) as a liable gene for spontaneous mutations in vitro

Document Type: Research Paper

Authors

1 Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Abstract

The apoptotic protease-activating factor 1 (Apaf-1) receives the death signal in the intrinsic or
mitochondrial pathway of apoptosis. Upon the releasing of cytochrome c from the
intermembrane space of mitochondria and binding to Apaf-1 molecules, a heptameric
apoptosome complex is formed and triggers the downstream cascade of caspases. Here, for the
first time we present spontaneous mutations and recombinations of the Apaf-1 gene and its
neighbouring sequences. We sequence 48 colonies containing pcDNA3.1 vector with
Nluc/Apaf1 and Cluc/Apaf1 obtained through the quick-change site-directed mutagenesis
method, transforming to DH5-α and XL10-Gold at two temperatures, 18 and 37ºC. In 21 of
these cases, we found 38 different mutations. Our data suggest that there is a direct relationship
between bacterial incubation temperatures and the number of unwanted spontaneous mutations.
During our experiment we found that the Apaf-1 gene is much less susceptible to spontaneous
mutations when it is transformed into XL10-Gold at 18 ºC . In contrast, a large number of
spontaneous mutations were found when the gene of interest transformed into DH5α at 37ºC .

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